Paramyxoviruses comprise a family of RNA viruses tropic for the human respiratory tract, that result in approximately 13 million infections per year. RSV, a major pathogen within the Paramyxovirus family, causes severe lung disease in young children, elderly adults, and immunocompromised individuals. To date, no effective vaccine for RSV presently exists despite the estimated 1 million deaths this virus causes annually in infants and children.
It has been found that most infected older children and adults restrict and eliminate RSV rapidly. However, young infants and children are susceptible to infection of bronchiolar and alveolar cells resulting in bronchiolitis, pneumonia or respiratory compromise which may result in death. Symptoms such as nasal discharge, fever, fatigue, deep cough, wheezing and shortness of breath are generally associated with RSV infections. Reinfections are common, especially in infants and young children.
Structurally, RSV is an enveloped, negative stranded RNA virus of the family Paramyxoviridae and of the genus pneumovirus. The two major envelope proteins are the G protein, which is believed to be responsible for attachment of the virus to the host cell membrane, and the fusion (F) protein, which is believed to be responsible for fusing the virus and cell membranes. Virus-cell fusion is a necessary step for infection. F protein is required for cell-cell fusion which is another way to spread the virus from an infected cell to an adjacent uninfected cell. Antibodies directed against these proteins do not confer significant immunity in humans.
The targets for RSV infections are generally the terminal bronchioles and alveoli in mammals which are lined by lung epithelial cells and alveolar macrophages. If these cells permit unrestricted RSV replication, viral burden progressively increases resulting in denudation of the airway and destruction of alveolar macrophages which defend the lung against other pathogens.
It has been found that most children can recover from RSV infection indicating that they can eliminate RSV. This implies that intrinsic cellular mechanisms, as yet undefined, may restrict virus replication. However, the reason why some children have progressive disease that can culminate in overwhelming infection and death is not yet known.
Although RSV has been extensively studied, there are no available effective vaccines to combat RSV. The lack of an effective vaccine for RSV suggests that strategies to augment intrinsic lung defenses against this virus could offer potential clinical benefits. Therefore, applicants examined whether differentiated human lung epithelial cells possess intrinsic mechanisms to restrict RSV replication, whether such intrinsic mechanisms could be augmented by anti-viral cytokines (e.g. IFN-.beta.), and whether anti-viral cytokines could be operative before induction of humoral or cell-mediated immune responses.
In this regard, applicants studied RSV replication in normal human lung epithelial cells transformed with an origin defective SV-40 vector, i.e. 9HTE (tracheal origin) and BEAS 2B cells (bronchiolar origin). In addition, applicants examined the non-transformed human A549 cells (alveolar epithelial origin) derived from an alveolar carcinoma. These differentiated lung epithelial cell lines offer an in vitro model with which insights into the molecular mechanisms that restrict RSV in human lung cells can be examined. In addition, the differentiated lung epithelial cell lines offer advantages over highly permissive cell lines, such as CV-1, HEp-2 cells, or HeLa cells, which lack characteristics of differentiated human lung cells.
Moreover, it is believed that alveolar macrophages may have an important role in restricting replication of respiratory viruses through their capacity to produce tumor necrosis factor a (hereafter referred to as TNF.alpha.). Local production of TNF.alpha. at the site of virus infection has previously been shown to restrict vaccinia virus replication (Sambhi, S.K., Kohonen-Corish, M. R. J., and L. A. Ramshaw. 1991. Local production of tumor necrosis factor encoded by recombinant vaccinia virus is effective in controlling viral replication in vivo. Proc Natl Acad Sci. 88:40254029). In vitro and in vivo studies have demonstrated that alveolar macrophages are permissive to RSV infection/replication and produce TNF.alpha. following RSV infection (Panuska, J. R., Hertz, M. l., Taraf, H., Villani, A., and N. M. Cirino. 1992. Respiratory syncytial virus infection of alveolar macrophages in adult transplant patients. Am. Rev. Respir. Dis. 145:934939).
Furthermore, it has been determined that TNF.alpha. restricts further RSV replication in alveolar macrophages through an autocrine mechanism. The intimate contact between alveolar macrophages and epithelial cells from terminal bronchioles and alveoli suggests that TNF.alpha. could potentially restrict RSV through paracrine mechanisms. However, the potential use of recombinant TNF.alpha. as a systemic antival therapy is limited by its toxicity.
Applicants therefore examined whether RSV could be transmitted between lung epithelial cells and alveolar macrophages. Applicants further examined TNF.alpha. binding to lung epithelial and CV-1 cells and whether these separate cell types expressed the 55 and 75 kDa TNF.alpha. receptor subtypes (Loetscher, H., Pan, Y. E., Lahm, H., Gentz, R., Brockhaus, M., Tabuchi, H., and W. Lesslauer. 1990. Molecular cloning and expression of the human 55 kd tumor necrosis factor receptor. Cell 61:351-359; Pennica, D., Lam, V. T., Mize, N. K., Weber, R. F., Lewis, M., Fendly, B. M., Lipari, M. T., and D. V. Goeddel. 1992. Biochemical properties of the 75-kDa tumor necrosis factor receptor. J. Biol Chem. 267:21172-211 78). The 55 kDa TNF.alpha. receptor mediates the anti-viral effects of TNF.alpha. in some (Wong, G. H. W., Tartaglia, L. A., Lee, M. S., and D. V. Goeddel. 1992. Antiviral activity of tumor necrosis factor (TNF.alpha.) is signaled through the 55-kDa receptor, type 1 TNF.J. Immunology 149:3350-3353), but not all cell types (Rothe, J., Lesslauer, W., Lotsher, H., Land, Y., Koebel, P., Kontgen, F., Althage, A., Zinkernagel, R., Steinmetz, M., and H. Bluethmann. 1993; Mice lacking the tumor necrosis factor receptor 1 are resistant to TNF mediated toxicity but highly susceptible to infection by Listeria monocytogenes. Nature 364:798-802).
In this regard, it is known to some degree that TNF.alpha. and IFN-.beta., individually, or synergistically, can restrict replication of both RNA and DNA viruses (Sen, G. C., and R. M. Ransohoff. 1993. Interferon-induced antiviral actions and their regulation. Adv. Virus Res. 42:57-101; Wong, G. H. W., Kamb, A., and D. V. Goeddel 1993. Antiviral properties of TNF. In B. Beutler, editor. Tumor Necrosis Factors: The Molecules and Their Emerging Role in Medicine.Raven Press. Ltd, New York, 371-381). IFN restricts RSV replication in lung fibroblast cell lines (Moehring, J. M., and B. R. Forsyth. 1971. The role of the interferon system in respiratory syncytial virus infections. Proc Soc Exp. Biol Med.138:1009-1014) but its effects on RSV replication in lung epithelial cells had not been previously examined. RSV induces low levels of IFN in only .about.50% of infected children (Hall, C. B., Douglas, R. G. Jr, Simons, R. L., and J. M. Geiman.1978. Interferon production in children with respiratory syncytial, influenza, and parainfluenza virus infections. J. Pediatr. 93:28-32) and does not induce IFN expression from macrophages in vitro (Roberts, N. J. Jr, Hiscon, J., and D. J. Signs. 1992. The limited role of the interferon system in response to respiratory syncytial virus challenge: analysis and comparison to influenza virus challenge. Microbial Pathogenesis.12:409414).
The Applicants have shown that RSV potently induces the cytokine surpressing inhibitor factor, termed IL-10, by infected human macrophages and epithelial cells which directly inhibits IFN production. (Hoffmann, S. P., Rebert, N. A., Panuska, J. R. 1995. Respiratory Syncytial Virus Induction of Lung Cell Expression of Interleukin 10: Implications For Incomplete Immunity. Am. J. of Resp. and Crit. Care Medicine 151:A774). Thus, RSV inhibits expression of anti-viral cytokines by resident lung cells.
However, IFN treatment of RSV infected children does improve their clinical course suggesting that exogenous IFN may augment intrinsic lung defenses against this virus (Sung, R. Y. T., Yin, J., Oppenheimer, S. J., Tam, J. S., and J. Lau. 1993. Treatment of respiratory syncytial virus infection with recombinant interferon alfa-2a. Arch Dis. Child 69:440-442).
Applicants therefore determined the effects of TNF.alpha. and IFN-.beta. alone, and in combination, on RSV infection and replication in lung epithelial cells as well as the highly RSV permissive CV-1 cell line. Moreover, applicants examined known mechanisms by which TNF.alpha. and/or IFN-.beta. restrict virus replication including inhibition of virus infection, cytostatic effects on cells, direct lysis of virus infected cells, or inhibition of protein synthesis which reflects induction of TNF.alpha./IFN-.beta. responsive anti-viral genes.
As shown below, applicants have found that IFN-.beta. restricts RSV replication in human lung epithelial cells, the natural target cells for this virus. Previously, delivery of ribavirin (1-beta-D-ribofuranosyl-1-1, 2, 4-triazole-3-carboxamide), a synthetic nucleotide that is administered intravenously by small particle aerosol for 12-20 h a day for approximately three (3) days was essentially the only anti-viral drug and/or treatment useful in RSV infections. However, ribavirin's clinical efficacy has been marginal and its potential teratogenic effects have limited its use. Nevertheless, it is used in .about.20,000 children per year at a cost of $3,000 per patient.
The present invention relates to the specific delivery of IFN-.beta. to the airway of a host susceptible or suffering from infection of RSV and other RNA respiratory viruses. Recent studies have indicated that INF-.beta. can be delivered by aerosol to volunteers without causing any harmful effects. (Halme, M., Maasilta, P., Mattson, K., Cantell, K. 1994. Pharmacokinetics and Toxicity of Inhaled Human Natural Interferon-Beta in Patients With Lung Cancer. Respiration 61:105-107). The invention has the ability to restrict RSV without inducing side effects that can be observed with parenterally administered IFN-.beta.. It also has the potential to restrict other RNA viruses which might be sensitive to cytokines.
These and other objects of the present invention will be more apparent from the discussion below.